In many transplantation-type situations, there is concern for differences between the allotype, especially the HLA type, of a cell source and the cell recipient. Antibodies against alloantigens can be induced by multiple blood transfusions, pregnancy, or during a prior graft rejection. Although these antibodies may be low titer, and difficult to detect, their presence in the blood of a potential recipient is indicative that a new graft with matching alloantigens will be rejected. The determination of the presence and specificity of antibodies against foreign HLA antigens is therefore clinically important for monitoring transplant patients. Detection assays may test for reactivity against a panel of antigens, as an initial broad screen (panel reactive antibodies, PRA testing), or may be specific for a single donor (donor specific crossmatch).
The standard technique for HLA typing and detection of anti-HLA antibodies is microlymphotoxicity, where serum containing antibodies is incubated with HLA antigen-expressing lymphocytes, then with complement. In some cases anti-human immunoglobulin is added to augment cell killing. The level of cytotoxicity is estimated by discriminating between dead and viable cells using various dyes. This method has numerous disadvantages: it is labor intensive, time consuming, requires isolation of cells, requires viable cells, is nonspecific for HLA, and requires a subjective evaluation. Flow cytometry may also be used but requires a large number of cells and expensive instrumentation.
It is therefore of interest to provide alternative techniques which can be performed simply, can be automated, do not share the shortcomings described above, provide a readily discernible result which is significant for the prognosis of graft acceptance, and comparable to data from existing tests.